Malignant primary brain tumors represent the most frequent cause of cancer death in children and young adults and account for more deaths than cancer of the kidney or melanoma. Glioblastoma (GBM) is uniformly lethal, and current therapy is non-specific and produces a median overall survival of <15 months. In contrast, immunotherapy promises an exquisitely precise approach, and substantial evidence suggests that T-cells can eradicate large, well-established tumors in mice and humans even when tumors reside within the brain. Chimeric antigen T-cell receptors (CARs) combine the variable region of an antibody with T-cell signaling moieties to confer T-cell activation with the targeting specificiy of an antibody and are not MHC-restricted. Additionally, co-stimulatory molecules, such as CD28 and 4-1BB, can be added to these constructs to improve T-cell expansion, survival, cytokine secretion, and tumor lysis. Clinical trials utilizing CARs have demonstrated their remarkable potential. However, severe adverse events and even patient deaths have occurred when these CARs have been directed against antigens shared by normal tissues. EGFRvIII is a tumor-specific mutation of the epidermal growth factor receptor that is expressed in GBMs and several other neoplasms. We have previously shown that EGFRvIII can be recognized by highly avid antibodies, so have developed human and murine CAR vectors that specifically recognize EGFRvIII inducing cytokine secretion and in vitro and in vivo tumor lysis. We have also demonstrated that an EGFRvIII-specific peptide (PEPvIII) contains the conformational epitope for EGFRvIII-specific antibodies used in these CARs. Using this peptide, we have shown that these cognate peptides are sufficient antidotes for CARs, suggesting a novel paradigm for reducing the target-specific toxicity of less tumor-specific CARs. Despite their potency, however, CARs still require host conditioning with lymphodepletion for efficacy and are still limited by being susceptible to inhibition by host immunosuppressive factors of which regulatory T-cells (TRegs) have been most frequently implicated. Similarly, while total body irradiation or non-therapeutic chemotherapy has been applied to optimize CAR therapy, it adds additional toxicity without direct anti-tumor efficacy. Our prior experience with TMZ demonstrates that, in addition to having direct clinical benefit in GBM, TMZ can potentiate anti-tumor immune responses directly related to the rebound homeostatic proliferation it induces. To address these issues, in this proposal, we will 1) Evaluate the risk of toxicity, utility of TMZ, and the requirements for efficacy of a tumor-specific, EGFRvIII-targeted CAR in a syngeneic, immunocompetent, orthotopic murine GBM model; 2) Determine if CD3-CD28-4-1BB CAR vectors naturally transfect and activate TRegs and dissect the role of CAR-secreted IL-2 in supporting the growth of intratumoral TRegs and effector T-cells; and 3) Conduct a Phase I clinical trial in TMZ-treated patients with EGFRvIII-expressing GBM to assess CAR safety, kinetics and function.